Vomeronasal organ and social factors affect urine marking by male mice

Adult CF-1 male mice cohabited for 15 days with three different females (EXPERIENCED) or remained physically isolated (INEXPERIENCED) prior to removal of their vomeronasal organs (VNX) or a SHAM procedure. Subjects were tested one month after surgery for urine marking in response to a stimulus male, a stimulus female, or no stimulus animal on the opposite side of a screened partition. VNX males exhibited a significant reduction in urine marking compared to SHAM males. EXPERIENCED VNX males deposited urine in amounts that were intermediate to SHAMs and INEXPERIENCED VNX males. These results suggest that deficits in urine marking behavior that result from loss of vomeronasal chemoreception may be ameliorated by prior sexual and/or social experience with females.     

Chemoinvestigatory and sexual behavior of male guinea pigs following vomeronasal organ removal

The vomeronasal organs of male guinea pigs were removed (VNX; n=10) or males experienced sham surgery (Sham; n=10). Subsequently a battery of chemosensory tests of investigatory responsiveness to conspecific urine was conducted. Additionally, male subjects were paired with female conspecifics for short and long periods and social and sexual behaviors were monitored. VNX males exhibited a depression in urine investigation and this depression became more profound following repeated testing and/or the passage of time. By 6.3 months following surgery, investigatory responsiveness to urine was practically eliminated. Maintenance of responsiveness to urine odors may require reinforcing input through the accessory olfactory system. In contrast to these effects on responsiveness to odors, VNX and Sham males were indistinguishable in their social and sexual behavior. These data indicate that male guinea pigs without a VNO: (1) Exhibit a depression of investigation of urine odors which is time dependent and which may involve an extinction-like process; (2) continue to discriminate classes of urine (e.g., urine from male vs urine from female conspecifics); and (3) exhibit normal sexual behavior. The vomeronasal organ in the male domestic guinea pig is apparently critical for the maintenance of normal responsiveness to sex odors but, in its absence, other sensory systems are capable of maintaining normal sexual behavior under conditions of laboratory testing.     

Mouse vomeronasal organ: effects on chemosignal production and maternal behavior

Adult male mice excrete a urinary chemosignal that accelerates puberty in females, whereas group-housed female mice excrete a urinary chemosignal that delays puberty in young females. We found that: (1) the excretion of the puberty-acceleration chemosignal by males persisted in the absence of the vomeronasal organs and (2) the puberty-delay chemosignal was not present in the urine of group-housed females whose vomeronasal organs had been surgically removed (VNX), but was present in the urine of group-housed females subjected to sham surgery (SHAM). These results suggest that in males, vomeronasal chemoreception does not affect the excretion of the puberty-acceleration chemosignal, but that in females, the vomeronasal organ receives chemosignals that influence the excretion of the puberty-delay chemosignal. Additionally, we found no difference between SHAM and VNX females in rates of conception, litter size, pup growth, pup recognition, or maternal behavior, indicating that normal maternal processes are expressed in the absence of an intact accessory olfactory system.     

Effects of vomeronasal organ removal on individual odor discrimination, sex-odor preference, and scent marking by female hamsters

Removal of the vomeronasal organ (VNX) did not eliminate the ability of female hamsters to discriminate between individual male's flank gland or urine odors in a habituation/discrimination task nor did it impair preference for male odors over female odors from a distance. Vomeronasal organ removal did reduce overall levels of investigation of flank gland odor in the habituation/discrimination task. Although VNX females did not show severe impairments in the frequency of either flank or vaginal marking in response to odors, they did show an abnormal pattern of marking. VNX females, unlike shams, did not flank mark more to female odors than to male odors, nor did they vaginal mark more to male odors than to female odors. Thus, the vomeronasal organ in female hamsters appears to be important for differences in scent marking toward male and female odors, but is not essential for discrimination of individual odors or for preferences for male over female odors.     

Vomeronasal organ removal and female mouse aggression: the role of experience

Removal of the vomeronasal organ (VNX) from female mice had little effect upon fecundity, nest building and pup retrieval. Surprisingly, VNX significantly affected aggression by lactating females. Normally, lactating females will attack and fight intruders placed into the home cage, especially if the intruder is an unfamiliar male. In Experiment 1, we determined that VNX prior to sexual experience totally eliminated this type of aggression. Intact females were highly aggressive, but much more so to unfamiliar than to familiar males. Copulation prior to surgery did not ameliorate the effects of VNX; again, none of the VNX females were aggressive (Experiment 2). In Experiment 3, attacks and fights by previously aggressive primiparous females also were eliminated or significantly suppressed following VNX. Even when the testing occurred later during the postpartum period and for extended periods of time, this same suppression of aggression held following VNX. We conclude that aggression by lactating female mice is under strict mediation by chemosignals detected by the vomeronasal organ.     

Removal of the vomeronasal organ disrupts the activation of reproduction in female voles

The reproductive system of female prairie voles remains quiescent in the absence of stimulation from males; however, chemosignals from males are capable of at least partially activating female reproduction. In other species, the vomeronasal system mediates some of the reproductive responses of females to males. We found that surgical removal of the vomeronasal organ (VNX) from adult female prairie voles impeded reproductive activation in response to pairing with stud males: ovarian and uterine weights of VNX females paired with stud males for 24 or 60 hours were significantly less than those of normal (NORMAL) or sham-operated (SHAM) females. Furthermore, 8 of 9 NORMAL, 10 of 13 SHAM, but only 4 of 9 VNX females paired with stud males for 60 hours mated. VNX females, however, were still able to use chemosensory cues to locate food. Behavioral observations of females encountering stud males were similar for VNX, SHAM and NORMAL females. We conclude that vomeronasal chemoreception may be a primary component of reproductive activation in female prairie voles, presumably by mediating neuroendocrine responses to chemosignals.     

The vomeronasal organ is required for the male mouse medial amygdala response to chemical-communication signals,as assessed by immediate early gene expression

Many species use chemical signals to convey information relevant to social and reproductive status between members of the same species (conspecific), but some chemical signals may also provide information to another species (heterospecific). Both of these types of complex chemical signals may be detected by the vomeronasal organ, which sends projections to the accessory olfactory bulb and on to the medial amygdala. Previous reports in hamster and mouse suggest that the medial amygdala sorts this complex chemosensory information categorically, according to its biological relevance (salience). In the present set of experiments, male mice having undergone vomeronasal removal surgery (VNX) or a sham-operation (SHAM) were exposed to conspecific (male and female mouse urine) or heterospecific (hamster vaginal fluid and worn cat collar) chemical stimuli. Similarly to our previous report with intact male mice [Samuelsen and Meredith (2009) Brain Res 1263:33–42], SHAM mice exhibit different immediate early gene (IEG) expression patterns in the medial amygdala dependent upon the biological relevance of the chemical stimuli. However, regardless of biological relevance, vomeronasal organ removal eliminates all responses in the medial amygdala to any of the chemical stimuli. Interestingly, VNX also disrupts the avoidance of (an unfamiliar) predator odor, worn cat collar. Here we show that the medial amygdala response to the tested chemical signals is dependent upon an intact vomeronasal organ.     

Vomeronasal and/or olfactory mediation of ultrasonic calling and scent marking by female golden hamsters.

The role of the olfactory and vomeronasal systems in mediating odor-stimulated ultrasonic calling, flank marking, and vaginal marking by female hamsters was investigated by selective lesions of either system. Removal of the vomeronasal organ resulted in reduced frequencies of ultrasonic calling by estrous and nonestrous females in response to conspecific odors but it had no influence on either scent marking behavior during the same tests. When tested immediately after separation from a male, ultrasonic calling was not reduced by vomeronasal removal, indicating that such surgery does not cause deficits in calling ability and that the vomeronasal organ specifically mediates odor-stimulated calling. Zinc sulfate treatment of the olfactory mucosa led to reduction in the frequency of ultrasonic calling, flank marking, and vaginal marking in response to conspecific odors. Females' ability to discover buried food was also impaired by this treatment. Thus, the stimulation of both scent marking behaviors due to the perception of conspecific odors appears to be mediated primarily by the olfactory system, whereas stimulation of ultrasonic calling is mediated by both olfactory and vomeronasal systems.     

Conspecific urine marking in male-female pairs of laboratory rats

Male-female pairs of rats were observed during social interactions for conspecific markings where an animal deposits urine on the body of a second animal. Injections of sodium fluorescein were used to change urinary color and provide a visible mark on the back of a conspecific. The general hypothesis tested in the three experiments was that a female would mark males differently dependent upon her hormonal status and upon the relative hormonal integrity of the males. Results of Experiment 1 were that males marked females more frequently than vice versa and female marking decreased during her estrus. Moreover, a diestrous female marked an aggressive male more than she marked a non-aggressive male (Experiment 2), and a diestrous female marked a castrated male receiving 800 micrograms testosterone propionate (TP) injections more copiously than she marked a castrated male receiving 200 micrograms TP injections (Experiment 3). Finally, aggressive and non-aggressive males marked females similarly, though the 800 micrograms TP males marked females more than the 200 micrograms TP males. These data were compared with findings from research on environmental marking in rodents, and an hypothesis was suggested that female rats use conspecific marking to identify and select males with preferred behavioral and endocrine characteristics.     

Urine marking in male mice: responses to natural and synthetic chemosignals.

The effects of natural chemosignals and their synthetic terpenic analogs on urinary marking in mice were investigated. The number and size of marking spots, and percentage of response and avoidance/counter-marking behavior, were measured for dominant and subordinate males. The males' marking behavior was found to differ significantly based on the type of stimulus sample used. Marking behavior in response to female urine was significantly altered by addition of synthetic analogs (farnesene isomers) to it. Both dominant and subordinate males avoided marking the section of the test area soiled with stimulus samples containing either natural or synthetic farnesenes. Differential responses of the dominant and subordinate male mice were observed.     

Olfactory but not vomeronasal mediation of scent marking by male golden hamsters

Although olfactory and vomeronasal projections to the olfactory bulb and to the rest of the brain are quite distinct, it is not clear how the functions of these two chemosensory systems differ. In these experiments we attempted to determine the roles of the main olfactory and vomeronasal sensory systems in mediating scent marking by male golden hamsters, a behavior known to be stimulated by odors of other hamsters. Lesions of the main olfactory mucosa by irrigation of the nasal cavity with a solution of zinc sulfate dramatically reduced the frequency of marking. The degree of reduction in marking was correlated with an independent test of ability to detect volatile odorants. In contrast, surgical removal of the vomeronasal organ had no effect on marking frequency. Thus scent marking by male hamsters is primarily mediated by the main olfactory system and does not require the presence of the vomeronasal system. These results are discussed in the context of a theory that stresses the importance of the main olfactory system in pattern recognition and, in particular, in many types of social recognition.     

The mother rat's vomeronasal organ is involved in detection of dodecyl propionate, the pup's preputial gland pheromone.

The aim of the present study was to analyze the respective roles of main and accessory olfactory systems in a particular pattern of Wistar rat maternal behavior: specific licking of pup's anogenital areas, a behavioral pattern crucial to pup survival--nonlicked pups cannot defecate, and die. Dodecyl propionate (DP), a chemical agent from rat pup's preputial glands was found to direct and regulate pup's anogenital licking. Primiparous dams underwent one of the following treatments: surgical removal of the vomeronasal organ (hereafter VNX), irrigation of nasal cavities with 5% ZnSO4 solution (ZN), both these treatments (VNX + ZN), surgical control (SC), saline irrigation control (SA), both these treatments (SC + SA), and normal control (N). Fewer pups (only 75%) of VNX dams survived to 15 days of age compared to controls or to females rendered anosmic by zinc-sulfate irrigation of the nasal cavity (>95% survival). Furthermore, pup growth (mass increase over time) was impaired by VNX and/or ZN treatment. Greater than normal amounts of time were spent licking pups' anogenital area by VNX females, although these dams did not spend as much time as normal females in close contact with either pups heads or filter papers anointed with dodecyl propionate. The experiments reported herein suggest that the chemosensory receptors in the vomeronasal organ of dams mediate MAGL responses to pup pheromone, dodecyl propionate, a pheromone from pups' preputial glands secretion, sustaining pups' anogenital licking by dams.     

Detection of estrus by male mice: Synergistic role of olfactory–vomeronasal system

In rodents, olfactory pathway comprises two distinct systems viz, the main olfactory and vomeronasal systems, both differing in anatomy, physiology and function. The precise role of the main olfactory/vomeronasal system in estrus detection is yet to be explored. Therefore, the present investigation was planned to elucidate the role of main olfactory and vomeronasal system in the estrus discriminating ability of male mice. Female urine samples of proestrus, estrus, metestrus, diestrus, ovarectomized, ovarectomized plus estrogen treated and prepubertal mice were used for the present study. In addition, the urine from intact, castrated and castrated with testosterone treated mice was also tested for odour preference by male mice. The male responders were categorized into three groups namely (a) normal, (b) ZnSO₄-irrigated and (c) vomeronasal organ (VNO)-ablated. The behavioural responses such as frequency and duration of visits to urine samples were carried out in a Y-maze apparatus to assess odour preference. The normal mice displayed more frequent visits to estrus urine samples than to non-estrus samples. In contrast, ZnSO₄-irrigated mice showed significant reduction in the frequency of visits towards estrus urine, whereas, the vomeronasal (VNO)-ablated mice did not show any noticeable preference. With regard to the duration of visits the VNO-ablated mice showed significant reduction in visiting time when compared to ZnSO₄-irrigated mice. This finding indicated that the main olfactory system (MOS) was involved primarily in the attraction from a distance, while the VNO played a major role in close proximity (pre-copulatory behaviour). The males spent less time with the urine of same-sex; however, the response was higher with castrated male urine which was reduced on testosterone treatment indicating that a specific odour in intact male causes aversive behaviour in male. This study provides support to the fact that volatile compounds could also be perceived by VNO, probably when the main olfactory system is in functional state. The study implies that the olfactory–vomeronasal system plays a synergistic role in the detection of estrus.     

Paradigm for comparing sexual behavior and scent marking in male gerbils

Gerbils are popular subjects for research on social behavior and olfactory communication, yet little has been reported about their sexual behavior, apparently because researchers have found it difficult to elicit mating in stiuations where it can be observed and quantified. Here we describe a paradigm for eliciting complete copulatory behavior in male gerbils. We also report on the relationship between scent marking and sexual behavior and indicate how several environmental variables affect these behaviors. Our data indicate that (1) scent marking and sexual behavior are related, i.e., males that display sexual activity mark more often than males that do not mate; (2) male gerbils are more likely to copulate late in the light phase of the light: dark cycle than early in the dark phase; (3) housing males in pairs or groups facilitates copulatory behavior; and (4) marking frequencies as assessed in the sex arenas are positively correlated with, but higher than, marking scores obtained in the openfield.     

Lack of an inhibitory effect of hyperprolactinemia on androgen-dependent marking.

An experiment was performed to determine if hyperprolactinemia (chronically elevated serum prolactin levels), which inhibits testosterone-activated male sexual activity, also affects other androgen-dependent behaviors. Thus defecation and urine marking in response to a novel environment were examined in sham-operated and pituitary-grafted (hyperprolactinemic) male rats that had been castrated or castrated and given subcutaneous testosterone implants. Both castration and pituitary grafting significantly inhibited defecation, with the inhibitory effects of hyperprolactinemia being most pronounced in the castrated non-testosterone-treated animals. In contrast, castration significantly reduced the amount of urine marking observed, but pituitary grafting was without effect on this behavior. Thus, although hyperprolactinemia may inhibit sexual activity through an antagonism of the activational effects of testosterone, these results suggest that this effect is specific to sexual behavior and does not involve a more generalized inhibition of the effects of testosterone on androgen-dependent behaviors.     

Dominant-subordinate relationships in castrated male mice bearing testosterone implants

This study tested the hypothesis that subordination in male mice is directly related to reduced levels of circulating testosterone. Several measures of aggressiveness and submissiveness were quantified during or after daily encounters between pairs of intact males, castrated males, or castrated males bearing testosterone implants that were designed to maintain plasma testosterone titers at levels slightly above those of intact males. Behavioral measures included the total amount of fighting that resulted from daily pairing, the tendency for subordinates to fight back, and the relative frequency of urine marking. In all these regards, pairs of testosterone-implanted, castrated males formed dominant-subordinate relationships that involved apparently normal submissive behavior (compared to intact control pairs). Thus we found no evidence that submissive behavior was directly related to a reduction in circulating testosterone titers.     

Scent marking in the musk shrew (Suncus murinus)

The hormonal regulation of scent marking was examined in male and female musk shrews. In males castration depressed flank, perineal, and throat marking frequencies, and a physiological dose of testosterone restored perineal and throat marking to intact levels. Females exhibited high levels of marking whether tested in a non-pregnant state, late in gestation, or when post-partum. Moreover, ovariectomy had no influence on marking behavior. Adrenalectomy, in addition to ovariectomy, caused a significant decline in two of the three marking behaviors examined, flank and perineal marking. These results demonstrate that these sexually monomorphic scent marking behaviors are under gonadal control in male musk shrews, but not in females. Furthermore, two of the three different types of scent marking examined in female musk shrews appear to require adrenal hormones. Throat marking in the female musk shrew apparently is not regulated by steroidal hormones.     

Influence of neonatal androgen on the display of territorial marking behavior in the gerbil.

The effect of the presence or absence of androgen during the neonatal period on territorial marking behavior in the Mongolian gerbil was studied. Scent marking frequency was 20-40 fold greater in males than in females. Gonadectomy depressed marking in males but not in females. Testosterone propionate (TP) therapy completely restored marking in male but increased marking in intact and ovariectomized females to only one fourth that in males. Genetic males castrated within 2 days postpartum did not mark more frequently than TP-treated females after TP treatment in adulthood. Genetic females given a single TP injection within 6 days postpartum marked at male levels after TP treatment in adulthood. Males castrated after Day 2 and females given TP after Day 6 displayed marking frequencies intermediate between normal male and female levels after TP treatment in adulthood. This study suggests that sexual dimorphism in territorial marking behavior is due to a sex difference in the competency to respond to androgen, and it appears that development of this competency occurs during the neonatal period and is regulated, at least in part, by androgen. The onset of this differentiation process occurs earlier in the male than in the female.     

Social stress effects on territorial marking and ultrasonic vocalizations in mice

Acute social defeat (SD) leads to transient and persistent physiological and behavioral changes. We examined the effects of acute SD on territorial urine marking and ultrasonic courtship vocalizations in DBA/2 male mice. Both behaviors are considered androgen dependent and are influenced by social status, with dominant mice displaying more of both behaviors. In Experiment 1, male mice that received SD displayed prolonged inhibition of territorial urine marking, relative to nondefeated control mice (NOSD). In addition, territorial marking increased with repeated tests. In Experiment 2, male mice that received 3 successive days of SD displayed fewer ultrasonic courtship vocalizations at 30 min. post-SD1 and 30 min. post-SD2, relative to NOSD mice. In Experiment 2, we also observed decreased territorial marking 4 weeks post-SD. In sum, SD induced prolonged inhibition of territorial marking, but had only transient effects on ultrasonic courtship vocalizations, suggesting that different mechanisms may mediate the maintenance of these behaviors.     

Effect of vomeronasal organ removal from male mice on their preference for and neural Fos responses to female urinary odors

Four experiments were conducted to determine whether vomeronasal organ (VNO) inputs in male mice mediate the rewarding properties of estrous female urinary odors. Sexually naive male mice with either an intact (VNOi) or lesioned (VNOx) VNO preferred to investigate female urine over water in Y-maze tests. Subsequently, VNOi males ran significantly more quickly and remained in nasal contact longer with estrous female urine than with male urine, whereas VNOx males investigated these odors equally. In home-cage habituation-dishabituation tests, VNOi males also investigated female urine significantly longer than did VNOx males, although both groups investigated female urine longer than other non-body odors. Finally, female urinary odors induced Fos in the nucleus accumbens core of VNOi males but not of VNOx males. Our results suggest that female urinary odors retain some incentive value in VNOx males. However, once direct nasal contact is made with female urine, VNO inputs further activate forebrain mechanisms that amplify the reward salience of this stimulus for the male mouse.